Adapter-ligated amplified DNA fragments are required for many applications in modern molecular biology techniques. For example, adapter-ligated amplicons constitute libraries of a target DNA intended for sequencing analyses. The target regions in a DNA sample are subjected to an amplification procedure, e.g. by a polymerase chain reaction (PCR). The amplicons resulting from the PCR are subsequently ligated to adapter-molecules. The adapter-ligated amplicons or fragments of the target DNA are then subjected to a sequencing reaction. The DNA adapter molecules may therefore be provided with a nucleotide sequence that is specific for the sequencing primers. The resulting population of adapter-ligated amplicons is referred to as a so-called library, especially an amplicon sequencing library.
Currently, there are several common methods to generate amplicon sequencing libraries.
One method uses conventional multi-step enzymatic reactions to ligate adapters to the amplicons. This method is e.g. disclosed in Illumina Manual, “Mate Pair Library Preparation”, 2009. Amplicons are generated with target-specific primers by PCR and then end-repaired. The end-repair step normally requires two enzymes: A polynucleotide kinase, normally the T4 polynucleotide kinase (PNK), that phosphorylates the 5′-terminus of the double stranded amplicon or PCR product, respectively, and enzymes with polymerase and exonuclease activities that make the ends of the PCR products blunt by either fill-in and trimming reactions. After the end-repair step, for sequencing on some platforms, such as those provided by Illumina®, an addition of adenine nucleotides is required by a so-called A-addition step. In this step an A-overhang is added to the 3′-terminus of the end-repaired PCR product, e.g. by Klenow fragment exo-, the large fragment of the DNA polymerase I having 5′→3′ polymerase activity but lacking both 3′→5′ exonuclease activity and 5′→3′ exonuclease activity. This is to generate a docking side for the sequencing adapters that have an overhang formed by thymidine nucleotides, i.e. a T-overhang. After the A-addition, the sequencing adapter can be ligated to the amplicon by a DNA ligase, normally the T4 DNA ligase. For other sequencing platforms such as those from Life Technologies®, e.g. Ion Torrent PGM or Proton, SOLid, the A-addition step is not needed and blunt-ended adapters are ligated directly to the end-repaired amplicons.
Another library preparation method is to use fusion primers that contain both a target specific sequence and a part of the adapter sequences. After the first round of the PCR and the amplification of the target-specific regions, a second round of the PCR can be performed with primers containing the complete adapter sequence to add the adapter sequence to the amplicon.
Document WO 2009/072972 discloses a method for the enzymatic ligation of dsRNA adapter molecules to a dsRNA molecule.
All these methods in the art are tedious and time-consuming. Moreover, the method using fusion primers could also post challenge on design of the suitable PCR primers that should amplify hundreds to thousands of amplicons in the same multiplex PCR reactions without generating extensive amount of non-specific products.
Against this background, it is an object of the present invention to provide for a method of preparing adapter-ligated amplicons where the problems associated with the prior art methods can be reduced or avoided. It is also an object of the present invention to provide for an improved method of preparing a sequencing library of a target DNA.
The present invention satisfies these and other needs.